Vitamin A metabolism gives rise to several active forms of retinoic acid (RA) which are involved in regulating gene expression during development, regeneration, and in the growth and differentiation of adult epithelial tissues [Maden, 1992; Chambon, 1995; Mangelsdorf, 1995].
Retinoic acid itself has been found to be useful therapeutically, notably in the treatment of cancers, including acute promyelocytic leukemia (APL), tumors of the head and neck, and skin cancer, as well as in the treatment of skin disorders such as the premalignancy associated actinic keratoses, acne, psoriasis and ichthyosis. Unfortunately, a progressive resistance to RA has been observed in the treatment of APL [Muindi, 1992] and this has been attributed to increased RA metabolism [see Muindi, 1992; and Muindi, 1994 for review]. Therapeutic administration of RA can result in a variety of undesirable side effects and it is therefore important to establish and maintain the minimal requisite doses of RA in treatment. For example, RA treatments during pregnancy can lead to severe teratogenic effects on the fetus. Adverse reactions to RA treatment also include headache, nausea, chelitis, facial dermatitis, conjunctivitis, and dryness of nasal mucosa. Prolonged exposure to RA can cause major elevations in serum triglycerides and can lead to severe abnormalities of liver function, including hepatomegaly, cirrhosis and portal hypertension.
Many laboratory studies have involved metabolites of RA, particularly the activities of all-trans and 9-cis RA metabolites. The mechanism of conversion between all-trans RA and 9-cis RA in vivo is unclear; the asymmetric distribution of these metabolites in developing embryos suggests that they may be preferentially sequestered or generated by tissue specific isomerases [Creech Kraft, 1994]. The normal balance of these metabolites is dependent upon rate of formation from metabolic precursors, retinol and retinaldehyde [Lee, 1990], and rate of catabolism. RA catabolism is thought to proceed through the formation of polar intermediates, including 4-hydroxy-retinoic acid (4-OH-RA) and 4-oxo-retinoic acid (4-oxo-RA) [Frolik, 1979]. It is unknown whether the 4-oxo- and 4-OH-metabolites are simply intermediates in the RA catabolic pathway or whether they can also have specific activities which differ from those of all-trans RA and 9-cis RA. Pijnappel et al. [Pijnappel, 1993] have shown that, in Xenopus, 4-oxo-RA can efficiently modulate positional specification in early embryos and exhibits a more potent ability to regulate Hoxb-9 and Hoxb-4 gene expression than all-trans RA. 4-oxo-RA has been found to bind to retinoic acid receptor-.beta. (RAR-.beta.) with affinity comparable to all-trans RA [Pijnappel, 1993] but poorly to RAR-.gamma. [Reddy, 1992], suggesting that this metabolite exhibits some receptor selectivity. 4-oxo-RA also binds to cellular retinoic acid binding protein (CRABP) but with an affinity slightly lower than that of all-trans RA [Fiorella, 1993]. Takatsuka et al. [Takatsuka, 1996] have shown that growth inhibitory effects of RA correlate with RA metabolic activity but it is unknown whether there is a causal relationship between production of RA metabolites and growth inhibition.
The generation of 4-oxo-RA and 4-OH-RA metabolites is believed to be a cytochrome P450 dependent process. This is because of an observed effectiveness of general P450 inhibitors such as ketoconazole and liarozole in inhibiting the production of these metabolites from RA [Williams, 1987; Van Wauwe, 1992; Van Wauwe, 1988; Van Wauwe, 1990]. In certain tissues (testis, skin, lung) and cell lines (NIH 3T3, HL 60, F9, MCF-7) RA metabolism can be induced by RA pretreatment [Roberts, 1979a & b; Frolik, 1979; Duell, 1992; Wouters, 1992; Takatsuka, 1996]. Studies involving targetted disruption of RAR genes in F9 cells suggest that RAR-.alpha. and RAR-.gamma. isoforms may play a role in regulating the enzymes responsible for this increased metabolism [Boylan, 1995].
It has recently been shown that 4-oxoretinol (4-oxo-ROL) can have greater biological activity than retinol. The 4-oxo-ROL is inducible by RA in F9 and P19 mouse teratocarcinoma cells [Blumberg et al., 1995; Achkar et al., 1996].
It is known that zebrafish fins regenerate through an RA sensitive process which utilizes many gene regulatory pathways involved in early vertebrate development [White, 1994; Akimenko, 1995a & b].
As far as the inventors are aware, cytochrome P450s involved in the metabolism of RA in extrahepatic tissues remain uncharacterized at the molecular level.